A family of viruses, named Circoviridae, found in a range of plant and animal species and commonly referred to as circoviruses, are characterized as round, non-enveloped virions with mean diameters from 17 to 23.5 nm containing circular, single-stranded deoxyribonucleic acid (ssDNA). The ssDNA genome of the circoviruses represents the smallest viral DNA replicons known. As disclosed in WO 99/45956, at least six viruses have been identified as members of the family according to The Sixth Report of the International Committee for the Taxonomy of Viruses (Lukert et al. 1995, The Circoviridae, pp. 166-168. In Murphy, et al. (eds.) Virus Taxonomy, Sixth Report of the International Committee on Taxonomy of Viruses, Arch. Virol. 10 Suppl.).
Animal viruses included in the family are chicken anemia virus (CAV); beak and feather disease virus (BFDV); porcine circovirus (PCV); and pigeon circovirus. PCV was originally isolated from porcine kidney cell cultures. PCV replicates in the cell nucleus and produces large intranuclear inclusion bodies (Murphy et al., 1999, Circoviridae p. 357-361, Veterinary Virology, 3rd ed. Academic Press, San Diego). There are currently two recognized types of PCV, PCV type 1 (PCV1) and PCV type 2 (PCV2). PCV1, isolated as a persistent contaminant of the continuous porcine kidney cell line PK-15 (ATCC CCL31), does not cause detectable cytopathic effects in cell culture and fails to produce clinical disease in pigs after experimental infection (Allan G., 1995, Vet. Microbiol 44: 49-64; Tischer et al., 1982, Nature 295:64-66; Tischer et al., 1986, Arch. Virol 91:271-276).
Unlike PCV1, PCV2 was initially associated with clinical diseases in the 1990s (Allan et al., 1998, J. Vet. Diagn. Invest. 10 (1), 3-10; Harding et al., 1998, J. Swine Health Prod. 6, 249-254; Le Cann et al., 1997, Piglet wasting disease, Vet. Rec. 141, 600; Segalés et al., 1997, Vet. Rec. 141, 600-601) and today it remains of economic importance in the swine industry worldwide.
Clinical signs of PCV2 infection manifest in a variety of ways including postweaning multisystemic wasting syndrome (PMWS), respiratory disease (porcine respiratory diseases complex, PRDC), porcine dermatitis and nephropathy syndrome (PDNS), enteritis, sometimes a combination of these in growing pigs, and reproductive failure in breeding age animals (Opriessnig et al., 2007, J. Vet. Diagn. Invest. 19 (6), 591-615). All these syndromes are often referred to as porcine circovirus diseases (PCVD) (Kekarainen, 2014, J. General Virology, 95, 1734-1742). There is evidence that PCV2 is ubiquitous and was present many years prior to the onset of clinical PCVD (Rose et al., Virus Research, 2012, 164, 78-89). Even though PDNS was reproduced in gnotobiotic pigs in the absence of PCV2 (Krakowka et al., 2008, Am. J. Vet. Res. 69, 1615-1622), pigs with clinical PDNS possess high levels of PCV2-specific antibodies, which are implicated in disease progression (Thomson et al., 2002, J. Vet. Med. A: Physiol. Pathol. Clin. Med. 49, 430-437; Wellenberg et al., 2004, Vet. Microbiol. 99 (3-4), 203-214). A more subtle manifestation of PCV2 infection is poor growth performance in apparently healthy herds (Horlen et al., 2008, J. Am. Vet. Med. Assoc. 232 (6), 906-912). More recently, a novel peracute syndrome has been described, termed acute pulmonary edema (APE), which appeared in vaccinated herds (Cino-Ozuna et al., 2011, J. Clin. Microbiol. 49 (5), 2012-2016). Unlike previously described syndromes, which are slow and progressive, APE is characterized by acute respiratory distress in apparently healthy animals followed by almost immediate death.
As many as thirteen open reading frames (ORFs) have been identified in the PCV2 genome. ORF1 (Meehan et al., 1998, Journal of General Virology 79(9):2171-2719; Meehan et al., 1997, J Gen Vir. vol. 78, pp. 221-227) comprises the nucleotides 398-1342 (GenBank Accession No. AF055392) and has the potential to encode a protein with a predicted molecular weight of 37.7 kD. ORF2 (Meehan et al., 1998; Meehan et al., 1997) comprises the nucleotides 1381-1768 joined to 1-314 (GenBank Accession No. AF055392) and may encode a protein with a predicted molecular weight of 27.8 kD. Further description of the PCV2 ORFs 1-13 may be found in U.S. Pat. Nos. 7,144,698, 7,192,594, 7,504,206, and 7,741,039. ORF1 gene encodes Rep and Rep′ proteins, which are related to virus replication. It is known that ORF2 gene encodes immunogenic structure capsid protein of PCV2, which is used to induce immune response in organisms.
Based on sequence analyses of PCV2 isolates, they can be further divided into three main genotypes (PCV2a-c). PCV2a and PCV2b have been documented worldwide (Segalés et al., 2008, Vet. Rec. 162 (26), 867-868), PCV2c has been documented in archived samples from Denmark (Dupont et al., 2008, Vet. Microbiol. 128 (1-2), 56-64).
Inactivated PCV2 vaccine is the most common commercially available PCV2 vaccine, such as CIRCOVAC (Merial), INGELVAC CircoFLEX (Boehringer Ingelheim Vetmedica), SUVAXYN PCV2 (Fort Dodge), which are based on inactivated PCV2. Published studies to date on PCV2 used either tissue homogenate or cultured virus derived from field isolates. Tischer et al. (1987, Arch Virol. 96:39-57) report that porcine kidney cells are stimulated to entry to the S phase in the cell cycle by D-glucosamine treatment. However, the treatment must be performed with caution because D-glucosamine is toxic for cell culture (see, Allan et al., 2000, J. Vet. Diagn. Investigation 12:3-14).
There is a remaining need for methods for culturing circovirus including, for example, PCV1, PCV2 and other circoviruses, such that circovirus in high yield is possible. Such methods would be advantageous, in particular for preparation of PCV2 antigens as vaccines directed against PMWS. The present invention addresses that need.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.